Christopher J. Ehrhardt

Phylogeny and molecular taxonomy of the Bacillus subtilis species complex and description of Bacillus subtilis subsp. inaquosorum subsp. nov.

The Bacillus subtilis species complex is a tight assemblage of closely related species. For many years, it has been recognized that these species cannot be differentiated on the basis of phenotypic characteristics. Recently, it has been shown that phylogenetic analysis of the 16S rRNA gene also fails to differentiate species within the complex due to the highly conserved nature of the gene, yet DNA-DNA hybridization values fall well below 70 % for the same species comparisons. As a complementary approach, we propose that phylogenetic analysis of multiple protein-coding loci can be used as a means to detect and differentiate novel Bacillus taxa. Indeed, our phylogenetic analyses revealed the existence of a previously unknown group of strains closely related to, but distinct from, Bacillus subtilis subsp. spizizenii. Results of matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses revealed that the group produces a novel surfactin-like lipopeptide with mass m/z 1120.8 that is not produced by the other currently recognized subspecies. In addition, the group displayed differences in the total cellular content of the fatty acids C(16 : 0) and iso-C(17 : 1)omega10c that distinguish it from the closely related B. subtilis subsp. spizizenii. Consequently, the correlation of these novel phenotypic traits with the phylogenetic distinctiveness of this previously unknown subspecies group showed that phylogenetic analysis of multiple protein-coding loci can be used as a means to detect and differentiate novel Bacillus taxa. Therefore, we propose that this new group should be recognized as representing a novel taxon, Bacillus subtilis subsp. inaquosorum subsp. nov., with the type strain NRRL B-23052(T) (=KCTC 13429(T)=BGSC 3A28(T)).

Archaeal and Bacterial Communities Respond Differently to Environmental Gradients in Anoxic Sediments of a California Hypersaline Lake, the Salton Sea

ABSTRACT Sulfidic, anoxic sediments of the moderately hypersaline Salton Sea contain gradients in salinity and carbon that potentially structure the sedimentary microbial community. We investigated the abundance, community structure, and diversity of Bacteria and Archaea along these gradients to further distinguish the ecologies of these domains outside their established physiological range. Quantitative PCR was used to enumerate 16S rRNA gene abundances of Bacteria, Archaea, and Crenarchaeota. Community structure and diversity were evaluated by terminal restriction fragment length polymorphism (T-RFLP), quantitative analysis of gene (16S rRNA) frequencies of dominant microorganisms, and cloning and sequencing of 16S rRNA. Archaea were numerically dominant at all depths and exhibited a lesser response to environmental gradients than that of Bacteria. The relative abundance of Crenarchaeota was low (0.4 to 22%) at all depths but increased with decreased carbon content and increased salinity. Salinity structured the bacterial community but exerted no significant control on archaeal community structure, which was weakly correlated with total carbon. Partial sequencing of archaeal 16S rRNA genes retrieved from three sediment depths revealed diverse communities of Euryarchaeota and Crenarchaeota, many of which were affiliated with groups previously described from marine sediments. The abundance of these groups across all depths suggests that many putative marine archaeal groups can tolerate elevated salinity (5.0 to 11.8% [wt/vol]) and persist under the anaerobic conditions present in Salton Sea sediments. The differential response of archaeal and bacterial communities to salinity and carbon patterns is consistent with the hypothesis that adaptations to energy stress and availability distinguish the ecologies of these domains.

Allochronic speciation, secondary contact, and reproductive character displacement in periodical cicadas (Hemiptera: Magicicada spp.): genetic, morphological, and behavioural evidence

Periodical cicadas have proven useful in testing a variety of ecological and evolutionary hypotheses because of their unusual life history, extraordinary abundance, and wide geographical range. Periodical cicadas provide the best examples of synchronous periodicity and predator satiation in the animal kingdom, and are excellent illustrations of habitat partitioning (by the three morphologically distinct species groups), incipient species (the year classes or broods), and cryptic species (a newly discovered 13‐year species, Magicicada neotredecim). They are particularly useful for exploring questions regarding speciation via temporal isolation, or allochronic speciation. Recently, data were presented that provided strong support for an instance of allochronic speciation by life‐cycle switching. This speciation event resulted in the formation of a new 13‐year species from a 17‐year species and led to secondary contact between two formerly separated lineages, one represented by the new 13‐year cicadas (and their 17‐year ancestors), and the other represented by the pre‐existing 13‐year cicadas. Allozyme frequency data, mitochondrial DNA (mtDNA), and abdominal colour were shown to be correlated genetic markers supporting the life‐cycle switching/allochronic speciation hypothesis. In addition, a striking pattern of reproductive character displacement in male call pitch and female pitch preference between the two 13‐year species was discovered. In this paper we report a strong association between calling song pitch and mtDNA haplotype for 101 individuals from a single locality within the M. tredecim/M. neotredecim contact zone and a strong association between abdomen colour and mtDNA haplotype. We conclude by reviewing proposed mechanisms for allochronic speciation and reproductive character displacement.

Use of Fatty Acid Methyl Ester Profiles for Discrimination of Bacillus cereus T-Strain Spores Grown on Different Media

ABSTRACT The goal of this study was to determine if cellular fatty acid methyl ester (FAME) profiling could be used to distinguish among spore samples from a single species (Bacillus cereus T strain) that were prepared on 10 different medium formulations. To analyze profile differences and identify FAME biomarkers diagnostic for the chemical constituents in each sporulation medium, a variety of statistical techniques were used, including nonmetric multidimensional scaling (nMDS), analysis of similarities (ANOSIM), and discriminant function analysis (DFA). The results showed that one FAME biomarker, oleic acid (18:1 ω9c), was exclusively associated with spores grown on Columbia agar supplemented with sheep blood and was indicative of blood supplements that were present in the sporulation medium. For spores grown in other formulations, multivariate comparisons across several FAME biomarkers were required to discern profile differences. Clustering patterns in nMDS plots and R values from ANOSIM revealed that dissimilarities among FAME profiles were most pronounced when spores grown with disparate sources of complex additives or protein supplements were compared (R > 0.8), although other factors also contributed to FAME differences. DFA indicated that differentiation could be maximized with a targeted subset of FAME variables, and the relative contributions of branched FAME biomarkers to group dissimilarities changed when different media were compared. When taken together, these analyses indicate that B. cereus spore samples grown in different media can be resolved with FAME profiling and that this may be a useful technique for providing intelligence about the production methods of Bacillus organisms in a forensic investigation.

Manifestations of hydrothermal discharge from young abyssal hills on the fast-spreading East Pacific Rise flank

Spectacular black smokers along the mid-ocean-ridge crest represent a small fraction of total hydrothermal heat loss from ocean lithosphere. Previous models of measured heat flow suggest that 40%–50% of oceanic hydrothermal heat and fluid flux is from young seafloor (0.1–5 Ma) on mid-ocean-ridge flanks. Despite evidence that ridge-flank hydrothermal flux affects crustal properties, ocean chemistry, and the deep-sea biosphere, few ridge-flank vent sites have been discovered. We describe the first known seafloor expressions of hydrothermal discharge from tectonically formed abyssal hills flanking a fast-spreading ridge. Seafloor manifestations of fluid venting from two young East Pacific Rise abyssal hills (0.1 Ma at 10°20′N, 103°33.2′W; 0.5 Ma at 9°27′N, 104°32.3′W) include fault-scarp hydrothermal mineralization and macrofauna; fault-scarp flocculations containing hyperthermophilic microbes; and hilltop sediment mounds and craters possibly created by fluid expulsion. These visible features can be exploited for hydrothermal exploration of the vast abyssal hill terrain flanking the mid-ocean ridge and for access to the subseafloor biosphere. Petrologic evidence suggests that abyssal hills undergo repeated episodes of transitory fluid discharge, possibly linked to seismic events, and that fluid exit temperatures can be briefly high enough to transport copper (≥250 °C).

Single cell profiling of surface carbohydrates on Bacillus cereus

Cell surface carbohydrates are important to various bacterial activities and functions. It is well known that different types of Bacillus display heterogeneity of surface carbohydrate compositions, but detection of their presence, quantitation and estimation of variation at the single cell level have not been previously solved. Here, using atomic force microscopy (AFM)-based recognition force mapping coupled with lectin probes, the specific carbohydrate distributions of N-acetylglucosamine and mannose/glucose were detected, mapped and quantified on single B. cereus surfaces at the nanoscale across the entire cell. Further, the changes of the surface carbohydrate compositions from the vegetative cell to spore were shown. These results demonstrate AFM-based ‘recognition force mapping’ as a versatile platform to quantitatively detect and spatially map key bacterial surface biomarkers (such as carbohydrate compositions), and monitor in situ changes in surface biochemical properties during intracellular activities at the single cell level.

Real-Time Observation of Antimicrobial Polycation Effects on Escherichia coli: Adapting the Carpet Model for Membrane Disruption to Quaternary Copolyoxetanes.

Real-time atomic force microscopy (AFM) was used for analyzing effects of the antimicrobial polycation copolyoxetane P[(C12)-(ME2Ox)-50/50], C12-50 on the membrane of a model bacterium, Escherichia coli (ATCC# 35218). AFM imaging showed cell membrane changes with increasing C12-50 concentration and time including nanopore formation and bulges associated with outer bacterial membrane disruption. A macroscale bactericidal concentration study for C12-50 showed a 4 log kill at 15 μg/mL with conditions paralleling imaging (1 h, 1x PBS, physiological pH, 25 °C). The dramatic changes from the control image to 1 h after introducing 15 μg/mL C12-50 are therefore reasonably attributed to cell death. At the highest concentration (60 μg/mL) further cell membrane disruption results in leakage of cytoplasm driven by detergent-like action. The sequence of processes for initial membrane disruption by the synthetic polycation C12-50 follows the carpet model posited for antimicrobial peptides (AMPs). However, the nanoscale details are distinctly different as C12-50 is a synthetic, water-soluble copolycation that is best modeled as a random coil. In a complementary AFM study, chemical force microscopy shows that incubating cells with C12-50 decreased the hydrophobicity across the entire cell surface at an early stage. This finding provides additional evidence indicating that C12-50 polycations initially bind with the cell membrane in a carpet-like fashion. Taken together, real time AFM imaging elucidates the mechanism of antimicrobial action for copolyoxetane C12-50 at the single cell level. In future work this approach will provide important insights into structure-property relationships and improved antimicrobial effectiveness for synthetic amphiphilic polycations.

An improved method for nanogold in situ hybridization visualized with environmental scanning electron microscopy

This paper is not subject to U.S. copyright. The definitive version was published in Journal of Microscopy 236 (2009): 5-10, doi:10.1111/j.1365-2818.2009.03207.x.

Separation of uncompromised whole blood mixtures for single source STR profiling using fluorescently-labeled human leukocyte antigen (HLA) probes and fluorescence activated cell sorting (FACS)

Analysis of biological mixtures is a significant problem for forensic laboratories, particularly when the mixture contains only one cell type. Contributions from multiple individuals to biologic evidence can complicate DNA profile interpretation and often lead to a reduction in the probative value of DNA evidence or worse, its total loss. To address this, we have utilized an analytical technique that exploits the intrinsic immunological variation among individuals to physically separate cells from different sources in a mixture prior to DNA profiling. Specifically, we applied a fluorescently labeled antibody probe to selectively bind to one contributor in a mixture through allele-specific interactions with human leukocyte antigen (HLA) proteins that are expressed on the surfaces of most nucleated cells. Once the contributors cells were bound to the probe, they were isolated from the mixture using fluorescence activated cell sorting (FACS)-a high throughput technique for separating cell populations based on their optical properties-and then subjected to STR analysis. We tested this approach on two-person and four-person whole blood mixtures where one contributor possessed an HLA allele (A*02) that was not shared by other contributors to the mixture. Results showed that hybridization of the mixture with a fluorescently-labeled antibody probe complimentary to the A*02 alleles protein product created a cell population with a distinct optical profile that could be easily differentiated from other cells in the mixture. After sorting the cells with FACS, genetic analysis showed that the STR profile of this cell population was consistent with that of the contributor who possessed the A*02 allele. Minor peaks from the A*02 negative contributor(s) were observed but could be easily distinguished from the profile generated from A*02 positive cells. Overall, this indicates that HLA antibody probes coupled to FACS may be an effective approach for generating STR profiles of individual contributors from forensic mixtures.

Integration of Gas Chromatography Mass Spectrometry Methods for Differentiating Ricin Preparation Methods

The investigation of crimes involving chemical or biological agents is infrequent, but presents unique analytical challenges. The protein toxin ricin is encountered more frequently than other agents and is found in the seeds of Ricinus communis, commonly known as the castor plant. Typically, the toxin is extracted from castor seeds utilizing a variety of different recipes that result in varying purity of the toxin. Moreover, these various purification steps can also leave or differentially remove a variety of exogenous and endogenous residual components with the toxin that may indicate the type and number of purification steps involved. We have applied three gas chromatography-mass spectrometry (GC-MS) based analytical methods to measure the variation in seed carbohydrates and castor oil ricinoleic acid, as well as the presence of solvents used for purification. These methods were applied to the same samples prepared using four previously identified toxin preparation methods, starting from four varieties of castor seeds. The individual data sets for seed carbohydrate profiles, ricinoleic acid, or acetone amount each provided information capable of differentiating different types of toxin preparations across seed types. However, the integration of the data sets using multivariate factor analysis provided a clear distinction of all samples based on the preparation method, independent of the seed source. In particular, the abundance of mannose, arabinose, fucose, ricinoleic acid, and acetone were shown to be important differentiating factors. These complementary tools provide a more confident determination of the method of toxin preparation than would be possible using a single analytical method.